Trivalent Radioisotope Bio-Targeted Radiopharmaceutical, Methods Of Preparation And Use

ABSTRACT

A targeted radiopharmaceutical comprising a targeting species chemically-bonded to a PCTA-chelated Q+3 trivalent radioactive ion of Formula Iis disclosed. Six of R1 through R7 are H and the seventh is a reacted functionality, Z, that forms the chemical bond with the targeting species, T. “g” is a number whose average value is 1 to about 12. X1, X2, and X3, are substituent groups that can coordinate to the Q+3 ion and/or help neutralize the ionic charge. Anion Y− is optionally present to balance the ionic charge. A pharmaceutical composition comprising a theranostic effective amount of a targeted radiopharmaceutical of Formula I in a pharmaceutically acceptable diluent is also contemplated, as are a method for treating and/or diagnosing a mammalian host having a disease, disorder or condition characterized by undesired angiogenesis, tumor growth and/or tumor metastasis.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. application Ser. No. 63/191,499and Ser. No. 63/191,506, both filed on May 21, 2021, whose disclosuresare incorporated herein by reference.

SEQUENCE LISTING

The sequence listing associated with this application is provided intext format in lieu of a paper copy and is hereby incorporated byreference into the specification. The name of the text file containingthe sequence listing is ______ the text file is ______ KB, was createdon ______, 2022; and is being submitted via EFS-Web with the filing ofthe specification.

BACKGROUND OF THE INVENTION

Radiopharmaceuticals typically contain a radioisotope attached to atargeting moiety or carrier. The radioisotope is carried to the targetby the carrier where it decays. The mode of isotope decay determines thetype of radiopharmaceutical.

Typically, gamma emitting isotopes are used to detect the fate of theconstruct and are used for diagnostic purposes. Constructs with particleemitters are preferred for therapy. Although beta-emitting radionuclideswere used previously, alpha-emitting radionuclides have shown excellentefficacy in recent years.

Alpha-emitting radionuclides are effective at killing cells in part dueto the short particle range and high linear energy transfer (LET). Potyet al. (J Nucl Med. 2018 June: 59(60):878-884) describe the use of alphaemitters for therapeutic radiopharmaceuticals.

The relatively long half-life of the alpha-emitting radionuclideactinium-225 (Ac-225) compared to other alpha emitters is one of thereasons that it has become popular as a therapeutic radioisotope for thetreatment of cancer. Clinical trials with constructs using the isotopehave shown excellent results. The about 10-day half-life is a good matchfor the in vivo biological half-life of monoclonal antibodies, and thefour alpha emissions produced by Ac-225 and its daughters wereresponsible for a high rate of tumor cell kill. However, the chemistrynecessary to attach Ac-225 to a targeting moiety was lacking.

Ac-225 ions exhibit a valence of +3, with a documented ionic radius of112 pm. Due to its lack of polarizability, Ac⁺³ is classified as a“hard” Lewis acid according to the Hard and Soft Acids and Bases (HSAB)[Pearson, J Am Chem Soc 1963, 85:3533-3539] theory and is thereforelikewise predicted to prefer “hard,” nonpolarizable, electronegativeLewis bases such as anionic oxygen donors. The hard/soft acid-baseproperties of a specific ion can be quantified using the concept ofabsolute (h) chemical hardness. The absolute chemical hardness (h) of anion is given by the equation (h)=(I−A)/2, where I is the ionizationenergy and A is the electron affinity of the species of interest. [Parrand Pearson, J Am Chem Soc, 1983; 105:7512-7516; and Pearson, Inorg Chem1988; 27:734-740.]

Absolute chemical hardness of Ac⁺³ and La⁺³ so calculated are 14.4 eVand 15.4 eV, respectively. Soft ions such as Au⁺, Ag⁺ and Cu⁺ exhibitabsolute chemical hardness values that's range from 5.7 to 6.3 eV,whereas conventional hard ions, like Sc⁺³ and Al⁺³ are characterized byabsolute chemical hardness values of greater than 24 eV. Thiele et al.,Cancer Biother Radio, 2018 33(8):336-348.

The large ionic size of Ac⁺³ is suited to large polydentate chelators ofhigh denticities, because most commonly used chelates for Ac(III) rangebetween 8-12 coordinate. Actinium is similar to other actinides and rareearth elements, and can undergo hydrolysis in solution in the absence ofa chelating agent to form [Ac(OH)_(3-x)]^(x-); the sub-picomolarconcentrations of Ac-225 cause the hydroxide species in turn to formradiocolloids that bind to surfaces such as reaction vessels.

Emission of multiple alpha-particles in the Ac-225 decay chain makesAc-225 a particularly effective isotope to kill cancer cells, yet alsomakes the directed delivery of the nuclide and its decay daughters achallenge. Due to the conservation of momentum, the emission of anenergetic alpha particle imparts a recoil energy to the daughter nucleusoften >100 keV, 1000 times larger than the binding energy for anychemical bond. This results in release of the daughter nuclide from thechelator of the original delivery vector. The subsequent redistributionof the alpha-emitting daughter nuclides in vivo can cause substantialharm to untargeted healthy tissues and reduce the therapeutic effect.

Davis et al., Nuc Med Biol 1999, 26(5):581-589 reported that limitedinformation exists regarding the behavior of Ac-225 in vivo. Preliminarystudies have evaluated Ac-225 complexed to citrate with respect totissue uptake, biodistribution, and tumor tropism in animal models.Previous studies using Ac-225 complexed to either of thepolyaminocarboxylate chelators, ethylenediamine tetraacetic acid (EDTA),or cyclohexyl diethylenetriaminepentaacetic acid (CHX-DTPA) showedvaried tissue tropism and elevated blood clearance compared withuncomplexed Ac-225.

Ac-225-CHX-DTPA-monoclonal antibody (Mab) complexes used to determinebiokinetic behavior on tumor-bearing nude mice showed successful invitro complexing but poor stability in vivo. Thus, whereas Ac-225 mayprove useful in radiotherapeutic models, information regardingpotentially effective chelators and the relative stability of suchAc-225 complexes in vivo is lacking.

A recent review article on Ac-225 radiopharmaceuticals, Robertson etal., Curr Radiopharm, 2018, 11(3):156-172, noted that the discovery of achelating agent that binds Ac(III) with sufficient stability and thatalso controls the release of its daughter nuclides remains a challenge.Moreover, limited Ac-225 global availability of and the absence of astable surrogate nuclide has limited the study of this isotope to ahandful of institutions around the world that have secured a reliableAc-225 supply.

The above review authors included the Davis et al. article, above, andnoted that biodistribution profiles over the course of 8 days for eachof the purified Ac-225-complexes were assessed by injecting 92 kBq (2.5mCi) of each complex and compared to the Ac-225-acetate biodistributionas a control.

Because uncomplexed Ac-225 accumulates predominantly in the liver withsmall amounts in the bone, kidney, and heart, high Ac-225 liver uptakeof a chelate indicates an unstable complex in vivo.Cyclohexyldiethylenetriamine-pentaacetic acid “a” isomer (CHX-A″-DTPA),and 1,4,7,10,13-pentaazacyclo-pentadecane-N,N′,N″,N′″,N″″-pentaaceticacid (PEPA) reduced liver uptake Ac-225 of the complex by more than 5.5times compared to Ac-225 acetate, and although the Davis et al. datasuggested —CHX-A″-DTPA to be the most effective tested chelator complexwith regard to its in vivo stability, the Robertson et al., reviewauthors wrote that “improvements can still be made to further reducenon-target tissue accumulation.” [Robertson et al., at page 164.]

As such, CHX-A″-DTPA provides inadequate chelation of Ac(III). Anotherimportant finding of the initial in vivo study on which Robertson et al.commented was that the maximum tolerated dose of Ac-225-CHX-A″-DTPA wasless than 185 kBq (5 mCi), because at doses of 185 kBq (5 mCi) orhigher, severe tissue damage was observed as early as 1 hourpost-injection (p.i.), which ultimately led to study animal death,causing 100% mortality by day 8 p.i.

Attachment of actinium to a targeting molecule was accomplished bySheinberg's research group (Sheinberg, Science 2001 Nov. 16;294(5546):1537-1540. doi: 10.1126/science.1064126). The chelator ofchoice was a bifunctional molecule based on DOTA. However, in theSheinberg group's report, a two-step method was used to obtain enoughAc-225 on the targeting moiety. In addition, yields based on Ac-225starting material were very low, less than 10% of the isotope wasincorporated into the targeting moiety. More than 90% of the isotope waswasted. Specific activities with this process ranged from about 50 to 70μCi per mg of antibody. Clearly, a one-step process with higher yieldswould be preferred.

Further studies of possible chelators by the Scheinberg research group[McDevitt et al., App. Radiat. Isot., 2002, 57(6):841-847] found that ofsix possible chelators studied, showed that only DOTA and1,4,7,10-tetraazacyclododecane-1,4,7,10-tetra-propionic acid (DOTMP)showed any complexation of Ac-225 after 2 hours at 37° C. withradiochemical yields (RCYs) of >99 and 78%, respectively. However,subsequent in vitro stability assays in serum suggested that the Ac-225DOTA complex was robust, remaining >90% intact after 10 days, whereasthe Ac-225-DOMTMP complex rapidly dissociated.

A two-step labeling process was again employed that requiredradiolabeling of the bifunctional DOTA-NCS ligand first, followed by mAbconjugation (pH 8.7, 37° C. for 52 minutes). Despite low overallradiochemical yields of only 9.8±4.5%, reasonable specific activity(4.1±2.6 GBq/g, or 0.11±0.07 Ci/g) was achieved that permittedpreclinical therapeutic studies. Low yields were attributed to the firstAc-225 labeling step of DOTA-NCS that required heating and,consequently, degradation of the isothiocyanate linker resulting in poormAb conjugation in the following step.

The Scheinberg group and co-workers [McGuire et al., J. Nucl. Med.,2014, 55(9):1492-1498] later reported a one-step process for preparationof Ac-225-DOTA-antibody constructs. That process proceeded in 2 Mtetramethyl ammonium acetate buffer (pH 7.5) with the addition ofL-ascorbic acid as radioprotectant to the addition of DOTA-antibodyconstruct and Ac-225⁺³ with a typical final reaction pH value of 5.8.Heating to 37° C. for 2 hours allowed a 10-fold increase inradiochemical yield (80%) compared to previous 2-step methods (6-12%),and resulted in the preparation of bioconjugates with up to 30-foldhigher specific activities (120 GBq/g compared to 3.7-14.8 GBq/g). Thehighest specific activity achieved was equivalent to 1 actinium forevery 25 antibodies.

US 2004/0067924 A1 (Frank) teaches the use of 12-membered macrocyclicamine-based polyacetate and polyphosphonate chelating agents forcomplexing Ac-225. DOTA-based chelating agents were found useful forchelating Ac-225.

Paragraph [0082] of that patent publication noted that the nitrobenzylgroup of one depicted DOTA chelant can be reduced to an aniline, whoseamine can be subsequently converted to an isothiocyanate to form abifunctional compound for linking to a targeting peptide antibody orother entity. A bifunctional analog of PCTA (below) was said could beprepared by attaching a linking group to one of the acetate carbons.

Chelating agents based3,6,9,15-tetraazabicyclo-[9.3.1]pentadeca-1(15),11,13-triene-3,6,9-aceticacid (PCTA) were mentioned in the text of the Frank application andbinding data with actinium were shown. The PCTA compound shown andutilized was not adapted for linkage to a targeting molecule such as apeptide or antibody other than by the possible use of one of thechelating carboxyl groups. No disclosure of a targeted construct usingPCTA was disclosed.

Yapp et al., Mol Imaging June 2013 12(4):263-272 reported on the use ofPCTA, DPTA and 1-oxa-4,7,10-triazacyclododecane-4,7,10-triacetic acid(Oxo-DO3A) for the chelation of Cu-64 [Cu (II)-64] for use in PET scanstudies of tumor vasculature. The chelates were bonded to the cyclictetrapeptide cyclic-(RGDyK) via benzylisothiocyanate linkages to theadded lysine of the cyclic peptide.

Another study, reported by Bryan et al., Cancer Biol Ther Jun. 15, 201111:12, 1001-1007, discussed the radioimmunotherapy and PET scan resultsof Cu-64 linked to an internalizing mAb and to a non-internalizing mAb,using 1,4,7,10-tetraazacyclo-dodecane-1,4,7,10-tetraacetic acid (DOTA)as the chelating agent in treating colon cancer tumors in xenograftedmice. Measuring the tumors daily, the results showed that the PET scanswere useful and that use of an internalizing antibody did not improvethe outcome of Cu-64 radioimmunotherapy.

An earlier one-step process was disclosed in Simón, WO 2011/011592 A1.This patent application teaches the preparation of a protein conjugatedwith chelators as a first step. After removal of excess chelating agent,the protein-chelated conjugate was reacted with the isotope. Again, aDOTA-based chelator was used for the work showing that the still currentthinking in the art was that DOTA-based chelators would be the best forAc-225.

The method in the Simon disclosure required the use of highconcentrations of acetate ion and a high chelator to antibody ratio(CAR). Starting reactions were conducted using a molar reactant ratio of100 chelators per antibody to yield a CAR number of 10-12.

It is desirable to produce high specific activity Ac-225 constructs withconjugates that have a lower CAR number. This is because as the CARnumber increases, the biological targeting of the antibody decreases.Thus, even though the one-step process is taught with Ac-225 and DOTAtype chelators, the CAR numbers required were high using DOTA-typechelating agents. Clearly there is a need for better chelating agentsfor preparing Ac-225 constructs.

The difficulties in using DOTA as a chelating agent for Ac-225 asdiscussed above notwithstanding, Thiele et al., Cancer Biother Radio,2018 33(8):336-348, as recently as 2018 used the phrase “DOTA: thecurrent gold standard” (at 340) for a section of their review. The lastsentence of those authors' DOTA section reads: “Collectively, theseshortcomings indicate that DOTA is not ideal for use in ²²⁵Ac-TAT [²²⁵Actargeted alpha therapy]applications, highlighting the need for moresuitable chelating scaffolds for ²²⁵Ac.”

The chemistry associated with attaching Ac-225 to targeting moieties hasbeen challenging for the users and writers. It is apparent that bettermethods of attaching Ac-225 to molecules are needed. The presentinvention helps address that need. Surprisingly, we have found thatPCTA-based chelating agents form stable chelates with Ac-225 under mildconditions and at lower CAR numbers than were previously reported whenusing DOTA, the prior “GOLD standard”.

Bi-213 is a radioactive decay product of Ac-225, whereas Bi-212 producedby the radioactive decay of lead-212 (Pb-212) after step-wise decay ofuranium-234 (U-234). The short half-life of Bi-212 and Bi-213 can limitthe application of these radionuclides in radionuclide therapy.

Bismuth isotopes, Bi-212 and Bi-213, are also candidates for use inradioimmunotherapy. Several preclinical studies have been publishedutilizing one, the other or both isotopes. Both have valences of +3, andas such tend to remain complexed after actinium has decayed.

Bi-212 has a half-life of about one hour and emits both alpha and betaparticles in an almost 1:2 ratio. Bi-213 has a half-life of about 45minutes and decays almost completely by beta emission to polonium-213,which then emits an alpha particle to form lead-209 as is shown in FIG.1 herein.

Illustratively, Park et al., Blood, 2020 116(20):4231-4239, reported apreclinical study in mice having xenografts of Ramos lymphoma that weretreated with anti-CD20 antibody fused to streptavidin followed by[²¹³Bi]DOTA-biotin. The treated mice with tumors exhibited marked growthdelays and mean survival times about four-times longer than untreatedcontrols. A review by Yong et al., AIMS Med Sci, 2021, 2(3):228-245,discussed recent work using Pb-212/Bi-212 in targeted a-particle therapy(TAT), such as work that utilized the chelator2-(4-isothio-cyanatobenzyl-1,4,7,10-tetraaza-1,4,7,10-tetra-(2-carbamonylmetyl)cyclododecane(TCMC) linked to a mAb, trastuzumab, that binds to HER2. A review byMulford et al., J Nucl Med 2005, 46(1 Suppl):199S-204S discusses severalTAT therapies that utilize one or the other of the above bismuthisotopes.

The labeling of biomolecules with precursor Pb-212 instead of Bi-212 orBi-213, as discussed in Yong et al., above, has the advantage ofobtaining a conjugate with a half-life of 10.6 hours, compared with of60 minutes for Bi-212 or 46 minutes for Bi-213. Previous attempts toprepare a potential in vivo generator with Pb-212 complexed by the DOTAchelator failed, because about 36% of Bi was reported to escape as aresult of the Pb-212 decaying via a beta particles to form Bi-212, whichwere not held by DOTA. It can be important that Bi-212 formed in thedecay of Pb-212 remain bound to the carrier because free bismuth ionslocalize in the kidneys. Bartos et al., J Radioanal Nucl Chem, 2013295:205-209.

Zirconium-89 is another useful radioisotope in that zirconium has avalence and the Zr-89 emits a gamma ray (909 keV) and also a positron atabout 397 keV, both emissions being useful in diagnostics. The half-lifeof Zr-89 is 3.3 days, which is similar to the circulation half-lives ofmany monoclonal antibodies used in medicine. Those isotopes have beenused in radiolabelling and evaluation of mAbs in positron emissiontomography (Immuno-PET). [Saleem et al., Sci World J 2014, Article ID269605, 9 pages.] The final decay product of Zr-89 is yttrium-89, astable non-radioactive isotope.

A further useful isotope in the present invention is indium-111. Indiumalso has a valence of +3, and In-111 has a half-life of about 2.8 days.Indium-111 decay provides gamma rays of 0.171 MeV and 0.245 MeV, thatcan be used in diagnostic scans such as single photon emission computedtomography (SPECT) imaging. In-111 decays to cadmium-111, which isnon-radioactive and stable.

The invention disclosed below teaches the using various targetingspecies and a single chelating agent for both therapeutic and diagnostic(theranostic) uses, providing a single chelator-linked targeting systemfor both uses. Such a theranostic has significant benefits indevelopment and manufacturing as the targeting species and chelationmanufacturing steps can be common with the labeling of the radioisotopebeing distinct. This provides some time and cost advantages indevelopment, toxicity studies with the unlabeled targeted-chelator,common stability and bulk drug substance.

SUMMARY OF THE INVENTION

This invention relates to the use of a chelating agent containing a12-membered macrocyclic amine with a pyridine ring imbedded in thestructure surprisingly that easily makes stable metal ligand complexeswith trivalent radioactive isotope ions such as Ac-225, Bi-212, Bi-213,Zr-89 and In-111, and also with a targeting species molecule to form aradiotherapeutic agent or a radiodiagnostic agent (or generically, aradiopharmaceutical agent). These radiopharmaceuticals can also bereferred to as radiotheranostic agents.

In one embodiment of the invention, the chelating agent is bonded to atargeting species molecule, permitting the chelation of the Q⁺³ ion tothat part of the molecule. The chelating agent is bonded to a part ofthe targeting molecule that does not interfere with the ability of thetargeting molecule to reach its target. The targeting species binds theradiopharmaceutical agent to cells that are to be killed or one or moreof whose presence, location, size and shape are to be determined.

More specifically, a targeting species is chemically-bonded to a PCTAchelator with its chelated trivalent radioactive isotope ion, Q⁺³, toform the theranostic radiopharmaceutical that has the general structuralformula shown below in Formula I and which, depending on the radioactiveisotope that is chelated can be used therapeutically to kill targetedcells or to bind to targeted cells to signal the one or more of thepresence, location, size or shape of the bound cells

In that chelator, the chelated Q⁺³ ion is a radioactive isotope having avalence of +3, six of R¹, R², R³, R⁴, R⁵, R⁶, and R⁷ are H, and theseventh contains a reacted functionality, Z, that forms the chemicalbond with the targeting species, T. X¹, X², and X³, are the same ordifferent substituent groups that can coordinate to that ion and/or helpneutralize the ionic charge of the chelated Q⁺³ ion such as trivalentAc-225, Bi-213, Bi-212, Zr-89 or In-111. “g” is a number whose averagevalue is 1 to about 12 that indicates the average number of chelatedPCTA-chelated trivalent radioactive ions, Q⁺³, per each molecule oftargeting species, T. An optional anion, Y⁻, can be present in an amountneeded to balance the ionic charge.

The chelation reaction with the Q⁺³ ion can be performed first followedby attachment to the targeting species molecule, T. This is referred toas a two-step process because the isotope is handled twice.Alternatively, the conjugation reaction (attaching the chelating agentto the targeting species) can be accomplished first followed byinsertion of Q⁺³ ion. This is called a one-step process as the isotopeis only handled once, and is preferred. ²²⁵Ac⁺³ is a preferred Q⁺³ ion.

A pharmaceutical composition is contemplated that comprises atheranostic effective amount of a targeted radiopharmaceutical ofFormula I dissolved or dispersed in a pharmaceutically acceptablediluent. Preferably, the pharmaceutically acceptable diluent is anaqueous liquid at ambient temperature and is adapted for parenteraladministration.

In one embodiment, that pharmaceutical composition is used in a methodfor treating a mammalian host having a disease, disorder or conditioncharacterized by undesired angiogenesis, tumor growth and/or tumormetastasis comprising administering to the host a targeted cell-killing(therapeutic) effective amount of the targeted radiopharmaceutical.

In further embodiments, a contemplated targeted radiopharmaceutical isused as a diagnostic agent. As such, the invention contemplates a methodfor assaying a mammalian host thought or known to have a disease,disorder or condition characterized by undesired angiogenesis, tumorgrowth and/or tumor metastasis by administering to the host a targetcell-binding effective amount of the targeted radiopharmaceuticalfollowed by scanning the host to detect and locate the radiation emittedby the bound targeted radiopharmaceutical.

BRIEF DESCRIPTION OF THE DRAWING

In the drawing forming a portion of this disclosure,

FIG. 1 shows the radioactive decay scheme from ²²⁹Th to stable ²⁰⁹Bi via²²⁵Ac in the development of the preparation of ²¹³Bi, showing theemission of four alpha particles (a) and four beta particles (b⁻) aswell as the half-life of each radionuclide in the decay scheme as shownboxes in which a numeral followed by a letter indicates the half-life,where d=day(s), h=hour(s), m=minute(s), ms=milliseconds, andms=microseconds, as are reported in Huang et al., Comput Math Method M,Vol. 2012, Article ID 153212, 6 pages;

DETAILED DESCRIPTION OF THE INVENTION

This invention relates to a targeted radiopharmaceutical that comprisesPCTA-chelated Q⁺³ ion chemically-bonded to a targeting species. Acontemplated targeted radiopharmaceutical of this invention has thegeneral structural formula shown below in Formula I

In that targeted radiopharmaceutical, Q⁺³ is a trivalent radioactiveisotope ion; six of R¹, R², R³, R⁴, R⁵, R⁶, and R⁷ are H and the seventhcontains a reacted linking functionality, Z, that forms a chemical bondwith the targeting species, T. The X¹, X², and X³ groups are the same ordifferent substituent groups that can coordinate to the Q⁺³ ion such as²²⁵Ac⁺³, ²¹²Bi⁺³, ²¹³Bi⁺³, ⁸⁹Zr⁺³ or ¹¹¹In⁺³ and help neutralize theionic charge of the chelated Q⁺³ ion. “g” is a number whose averagevalue is about 1 to about 12 that indicates the average number ofchelated PCTA-chelated trivalent radioactive ions, Q⁺³, per eachmolecule of targeting species, T. An optional anion, Y⁻, can be presentin an amount needed to balance the ionic charge.

Illustrative targeted radiopharmaceutical chelates are illustrated inFormulas Ia, Ib, Ic and Id below without depicting a specific targetingspecies, the number of chelates bonded to each target species, or areacted linking functionality, Z. The two bismuth isotopes (²¹²Bi⁺³ and²¹³Bi⁺³) are written together as ^(212/213)Bi⁺³ for convenience.

Actinium-225 is a preferred radiopharmaceutical isotope because it has ahalf-life of almost ten days and as shown in FIG. 1 , decays to releasefour alpha-particles and three beta-particles to form bismuth-209, astable isotope. One of the daughter decay products from Ac-225 isBi-213, and the final decay product is Bi-209, which is not radioactiveand is stable.

Bismuth-212 is a decay product of lead-212. Once obtained, thebismuth-212 can be separated from the lead-212 and complexed with anappropriately linked PCTA to form a chelated Q⁺³ ion chemically-bondedto a targeting species. Bi-212 ultimately decays to lead-208, which isnot radioactive and is stable.

Some versions of the chelating agent are referred to as pyridine-based12-membered tetraaza-macrocyclic ligands or PCTA (First published: 2Apr. 2019chemistry-europe.onlinelibrary-.wiley.com/-doi/abs/10.1002/ejoc.201900280.

In one embodiment, the reacted functionality, Z, is selected from thegroup consisting of one or more of a reacted Michael reaction acceptor,a reacted isocyanato group, a reacted carboxyl group, and a1,4-disubstituted-1,2,3-triazine formed by the reaction of an azide andan alkyne. A reacted isothiocyanate [—NH—C(═S)—NH—; a thiourea] is onepreferred reacted functionality.

The number of chelators bonded per antibody molecule is an averagenumber because some antibody molecules in a given composition do notreact whereas others do react. Average numbers of chelators bonded perantibody molecule are 1 to about 12, preferably about 3 to about 12, andmore preferably about 8 to about 10 when an isothiocyanate group isbeing bonded to an intact antibody. Where a paratope-containing portion(or antigen-binding fragment) thereof is the targeting species, thenumber of PCTA chelators per targeting species molecule tend to be fewersuch as about 1 to about 5 as there are fewer lysine amino groups withwhich the isothiocyanate group can react when the two pairs of CH2 andCH3 portions of the heavy chain are absent.

An illustrative chelator reacted functionality, Z, prior to reaction canbe a Michael reaction acceptor such as maleimide can link up to about 8chelators to a reduced intact antibody thiol groups. Of course, withsmaller targeting species such as a the peptidomimetic cyclic-(RGDyK)discussed hereinafter has only one amine that can bond to the chelator,thereby limiting the number of radiopharmaceutical chelates to which itcan be linked.

A Michael reaction acceptor contains an a,b-unsaturated carbonyl groupthat can react with a nucleophile such as an amine or a mercaptan.Illustrative Michael reaction acceptor groups include acryloyl,methacryloyl and maleimido groups.

Precursors for the formation of the 1,4-disubstituted-1,2,3-triazine, anazide and an alkyne can be present, one each, on either the pre-reactedfunctionality of the chelator or on the targeting species. The couplingreaction can be catalyzed by a copper(II) ion or by irradiation with UVlight.

The targeting species, T, is selected from the group consisting of oneor more of a chemically-bonded antibody or paratope-containing portionof an antibody, a chemically-bonded hormone, a chemically bondednon-antibody protein, a chemically-bonded cytokine, a chemically bondedaptamer, a chemically bonded oligonucleotide, a chemically-bondedcytokine, a straight chain or cyclic oligopeptide or peptide mimetic,and a straight chain or branched chain oligosaccharide. A monoclonalantibody (mAb) or a paratope-containing portion thereof is a preferredtargeting group, and a humanized monoclonal antibody or aparatope-containing portion thereof is particularly preferred.

The X¹, X², and X³ groups are the same or different substituent that isa functional group useful for chelation that can coordinate to the Q⁺³ion and/or help neutralize the ionic charge of the targetedradiopharmaceutical. Exemplary X substituents include a —(CH₂)_(n)CO₂Mgroup, a phosphonic acid (—PO₃M₂) group and half-esters thereof, as wellas carboxamides —(CH₂)_(n)CONH₂, and —(CH₂)_(n)CH₂NR¹⁰R¹¹ primary,secondary or tertiary amines where R¹⁰ and R¹¹ are the same or differentH or C₁-C₄ alkyl. In such a substituent, M is a proton (H⁺), an ammoniumion or an alkali metal ion. It is preferred that each of the X¹, X², andX³ groups is the same, and more preferably each is a —COOM group. “n” iszero or 1, preferably zero so that an X group is —CO₂M. It is to beunderstood that once in an aqueous composition such as a buffer, thecationic M is likely exchanged for another cation present in the aqueouscomposition.

A preferred chelator is referred to in the art as PCTA. The chemicalformula for a particularly preferred form of PCTA is the(4-isothiocyanato-phenyl)methyl derivative that enables the chelator tobe bifunctional, and is shown in Formula II, below, where M is as beforedescribed.

The chelator of Formula II is commercially available from MacrocyclicsInc. (Dallas, Tex.) under the designation p-SCN-Bn-PCTA.

Targeting Species

Antibodies are one group of preferred targeting species molecules in oneaspect of the invention because many bind to cell surface antigens ofundesirable cells in the body such as cancer cells. Once bound, theantibody and its bonded chelated Q⁺³ ion can be taken into the unwantedcell (cell to be treated) at which time the Q⁺³ ion such as Ac-225 orone of its daughter atoms such as ²¹³Bi⁺³ can decompose to release itscytotoxic alpha particle within the unwanted cell. The particularcombination of a Q⁺³ ion with a PCTA chelator forms particularly stablechelation products as compared to those formed using DOTA as thechelator with a Q⁺³ ion. As a consequence, there is a greaterconcentration of radioisotope at the target cell and a lowerconcentration of radioisotope elsewhere in the recipient body than whena chelator such as DOTA is utilized.

An illustrative list of monoclonal antibodies for use as a targetingspecies is provided in the table below. Most on the list are human orhumanized and are approved for being used in treating a human, whereasothers are mouse antibodies. It is to be understood that this list isonly illustrative with approximately 80-90 possibly useful monoclonalantibodies awaiting approval by the U.S. FDA for use in humans.

Commercial or mAb Name Other Name Target (Anti-) Source LintuzumabSGN-33 CD33 PDL BioPharma Gemtuzumab Mylotarg ™ CD33 Pfizer RituximabRituxan ® CD20 Genentech Tosityomab Bexxar ® CD20 Corixa Corp CetuximabErbitux ® EGFR Eli Lilly & Co. ATN-615 — uPAR Monopar Therapeutics Inc.ATCC Accession #PTA-8192 ATN-658 — uPAR Monopar Therapeutics Inc. ATCCAccession #PTA-8191 MNPR-101 — uPAR Monopar Therapeutics Inc. HumanizedATN-658 Brentuximab Adcetris ® CD30 Seattle Genetics TrastuzumabHerceptin ® HER2 Genentech Adalimumab Humira ® TNFa Abbott DaratumumabDarzalex ® CD38 Janssen Biotech Bevacizumab Avastin ® VEGF-A GenentechRosopatamab- — PSMA Convergent Therapeutics, Inc.; ATCC HB- 225Ac J59112126 E99 — PSMA Convergent Therapeutics, Inc.; ATCC HB- 12101 J415 —PSMA Convergent Therapeutics, Inc.; ATCC HB- 12109 J533 — PSMAConvergent Therapeutics, Inc.; ATCC HB- 12127 Atezolizumab Tecentriq ®PD-L1 Genentech Avelumab Bavencio ® PD-L1 Pfizer Basiliximab Simulect ®CD20 Novartis Canakinumab Haris ® IL1b Novartis Cemiplimab Libtayo ®PD-1 Regeneron Pharmaceuticals Cetuximab Erbitux ® EGFR Imclone/LillyDenosumab Prolia ® RANK Ligand Amgen Dinutuximab Garziba ® GD2 UnitedTherapeutics Durvalumab Imfinzi ® PD-L1 AstraZeneca Eculizumab Soliris ® C5 Alexion Elotuzumab Empliciti ® SLAMF7 Bristol-Myers Squibb,AbbVie Golimumab Simpopni ® TNFa Johnson & Johnson Infliximab Remicade ®TNFa Johnson & Johnson Ipilimumab Yervoy ® CTLA-4 Bristol-Myers SquibbIsatuximab Sarclisa ® CD38 Sanofi Genzyme Mogamulizumab Poteligeo ® CCR4Kyowa Kirin Motavizumab Numax ® RSV Meddimune Natalizumab Tysabri ®A4-Integrin Biogen Idec Necitumumab Portrazza ® ® EGFR Eli Lilly & Co.Nivolumab Opdivo ® PD-1 Bristol-Myers Squibb Obinutuzumab Gazyva ® CD20Genentech/Roche Ofatumumab Azerra ® CD20 Genmab Olaratumab Lartruvo ®PDGFRa Eli Lilly & Co. Omalizumab Xolair ® igE Genentech/RochePalivizumab Synagis ® RSV MedImmune Panitumumab Vectibix ® EGFR AmgenPembrolizumab Keytruda ® PD-1 Merck & Co. Pertuzumab Perjeta ® HER2Genentech/Roche Ramucirumab Cyramza ® VEGFR2 Eli Lilly & Co. RanibizumabLucentis ® VEGF Genentech/Roche Raxibacumab ABThrax ® B. anthrasisGlaxoSmithKline Tocilizumab Actemra ® Anri-IL6R Chugai/Roche UstekinumabStelara ® II-12/23 Johnson & Johnson

The mAb used illustratively herein designated mAb MNBR-101 is ahumanized version of mouse mAb ATN-658, whose hybridoma has ATCCAccession Number BTA-8191, disclosed and claimed in U.S. Pat. No.8,101,726. Mouse mAb ATN-615 that is also disclosed and claimed in U.S.Pat. No. 8,101,726, is secreted by a hybridoma that has ATCC AccessionNumber BTA-8192.

These mAbs specifically bind to (immunoreact with) the binary complexreferred to as uPA-uPAR; i.e., urokinase plasminogen activator (uPA) andits cell surface receptor, uPAR, as well as to uPAR at a locus that doesnot interfere with formation of the binary complex. U.S. Pat. No.8,101,726 notes that expression of uPA and uPAR has been demonstrated innumerous tumor types.

The mAb MNPR-101 paratopic amino acid residue sequence (CDR;complementarity determining region; variable region) binds to itsuPA-uPAR antigen very similarly to the binding of mAb ATN-658 Table 3,hereinafter). The heavy chain constant regions (CH1, CH2 and CH3) arethose of a human IgG1 antibody.

Humanization of ATN-658 to prepare MNPR-101 utilized the Xoma HE™synthesis platform that utilizes the human antibody amino acid residuesequences reported in Wu and Kabat, 1992 Mol. Immunol., 29(9):1141-1146(hereinafter Kabat) combined with the sequences of the variable regionsof the antibody to be humanized to form one or more consensus sequences.There are several steps in this process:

(1) Human Engineer™ (HE™) the ATN-658 Light and Heavy chains using theXOMA Corp. (Emeryville, Calif.) proprietary HE™ method to generate thelow risk and low plus moderate risk HE™ variants;

(2) HE™ Variable (V) region sequences codon optimization, energyminimization and gene synthesis;

(3) Clone the 4 HE™ V regions into XOMA's proprietary transientexpression vectors which contain human gamma-1 and kappa constant regionmodules;

(4) Transiently express the HE™ variants;

(5) Purify the humanized antibodies and characterize them for purity andendotoxin; and

(6) Verify the affinity of the 4 HE™ variants.

The phrase “low risk” discussed above and hereinafter relates to whethera mouse-to-human amino acid residue change results in a major reductionin therapeutic immunogenicity with little chance of affecting bindingaffinity. The second phrase “high risk” relates to modifying positionsat which a mouse-to-human amino acid residue change results in adegradation or abolition of binding activity with little or no actualreduction in therapeutic immunogenicity.

Humanization of ATN-658 to create mAb MNPR-101 using the Xoma HE™platform was performed pursuant to the “low risk”, “moderate risk” and“high risk” substitutions suggested in the following publications,patents and application: 1) WO 93/11794 “Methods and materials forpreparation of modified antibody variable domains and therapeutic usesthereof”; 2) U.S. Pat. No. 5,766,886 “Modified antibody variabledomains”; 3) U.S. Pat. No. 5,770,196 “Modified antibody variable domainsand therapeutic uses thereof”; 4) U.S. Pat. No. 5,821,123 “Modifiedantibody variable domains”; 5) U.S. Pat. No. 5,869,619 Modified antibodyvariable domains, and 6) Studnicka et al. 1994 Protein Eng 7:805-814,all of whose disclosures are incorporated by reference.

Further details related to the preparation of mAb MNPR-101 are discussedin the Examples hereinafter.

The term “antibody” is meant to include both intact immunoglobulin (Ig)molecules as well as fragments and derivative thereof, that can beproduced by proteolytic cleavage of Ig molecules or engineeredgenetically or chemically. Paratope-containing portions or fragmentsinclude, for example, Fab, Fab′, F(ab′)₂ and Fv, each of which iscapable of binding antigen. These fragments lack the Fc fragment ofintact antibody (Ab) and have an additional advantage, if usedtherapeutically, of clearing more rapidly from the circulation andundergoing less non-specific tissue binding than intact antibodies.Papain treatment of Ig's produces Fab fragments; pepsin treatmentproduces F(ab′)₂ fragments. These fragments can also be produced bygenetic or protein engineering using methods well known in the art.

A Fab fragment or portion is a multimeric protein consisting of theportion of an Ig molecule containing the immunologically active portionsof an Ig heavy (H) chain and an Ig light (L) chain covalently coupledtogether and capable of specifically combining with antigen. Fabfragments are typically prepared by proteolytic digestion ofsubstantially intact Ig molecules with papain using methods that arewell known in the art. However, a Fab fragment can also be prepared byexpressing in a suitable host cell the desired portions of Ig H chainand L chain using methods well known in the art. A F(ab′)₂ fragment is atetramer that includes a fragment of two H and two L chains.

The Fv fragment is a multimeric protein consisting of theimmunologically active portions of an Ig H chain variable (V) region(V_(H)) and an Ig L chain V region (V_(L)) covalently coupled togetherand capable of specifically combining with antigen. Fv fragments aretypically prepared by expressing in suitable host cell the desiredportions of Ig V_(H) region and V_(L) region using methods well known inthe art.

Single-chain antigen-binding protein or single chain Ab, also referredto as “scFv,” is a polypeptide composed of an Ig V_(L) amino acidresidue sequence tethered to an Ig V_(H) amino acid residue sequence bya peptide that links the C-terminus of the V_(L) sequence to theN-terminus of the V_(H) sequence. In a preferred embodiment, the Ab is amouse monoclonal antibody (mAb) designated ATN-615 (Creative Biolabs,Inc., Shirley, N.Y.) or ATN-658 (hybridoma B cell:ATCC PTA-8191;Manassas, Va.), both of which are IgG1 antibodies.

An Ab of the present invention can be produced as a single chain Ab orscFv instead of the normal multimeric structure. Single chain Absinclude the hypervariable regions from an Ig of interest and recreatethe antigen binding site of the native Ig while being a fraction of thesize of the intact Ig (Skerra et al., Science, 1988 240:1038-1041;Pluckthun et al. Methods Enzymol 1989 178:497-515; Winter et al., Nature1989 349:293-299); Bird et al., Science 1988 242:423-426; Huston et al.Proc. Natl. Acad. Sci. USA 1988 85:5879-5883; Jost et al., J Biol Chem.1994 269:26267-26273; U.S. Pat. Nos. 4,704,692, 4,853,871, 4,946,778,5,260,203, and 5,455,030).

DNA sequences encoding the V regions of the H chain and the L chain areligated to a linker that encodes a sequence of at least about 4 aminoacid residues (typically small neutral amino acids). The protein encodedby this fusion permits assembly of a functional variable region thatretains the specificity and affinity of the original Ab.

A different type of single chain Ab is an antibody induced in a camelidsuch as a dromedary, llama, alpaca, of vicuna. These animals producesingle heavy-chain-only antibodies (HcAbs) that have a variable regionand a constant region, with many unique properties such as small size,excellent solubility, superior stability, quick clearance from blood,and deep tissue penetration. The nomenclature of “nanobody” originallyadopted by the Belgian company Ablynx® due to its nanometric size andmolecular weight of less than about 15 kDa. Sanofi's affiliate AblynxN.V. is the holder worldwide of the NANOBODY® trademark.

The antigen-binding capacity of nanobodies, however, remains similar tothat of conventional antibodies for the following reasons. First, thecomplementarity-determining region 3 (CDR3) of nanobodies is similar oreven longer than that of human VH domain (variable domain of heavyimmunoglobulin chain). The former consists of 3 to 28 amino acids (AAs),whereas the latter only 8 to 15 AAs. As therapeutic agents, nanobodiesenable a targeted therapy by lesion-specific delivery of drugs andeffector domains, thereby improving the specificity and efficacy of thetherapy. [Bao et al., EJNMMI Res (2021) 11:6.] Humanized versions ofmonoclonal nanobodies are typically prepared in a manner similar to thatused for the preparation of double-chained mAbs, except that theytypically require fewer steps.

Human genes that encode the constant (C) regions of the chimericantibodies of the present invention can be derived from a human fetalliver library or from any human cell including those which express andproduce human Igs. The human C_(H) region can be derived from any of theknown classes or isotypes of human H chains, including g, m, a, d or e,and subtypes thereof, such as G1, G2, G3 and G4.

Because the H chain isotype is responsible for the various effectorfunctions of an Ab, the choice of C_(H) region will be guided by thedesired effector functions, such as complement fixation, or activity inAb-dependent cellular cytotoxicity (ADCC). Preferably, the C_(H) regionis derived from g1 (IgG1), g3 (IgG3), g4 (IgG4), or m (IgM).

The human C_(L) region can be derived from either human L chain isotype,k or 1.

Genes encoding human Ig C regions are obtained from human cells bystandard cloning techniques [Sambrook, J. et al., Molecular Cloning: ALaboratory Manual, 2nd Edition, Cold Spring Harbor Press, Cold SpringHarbor, N.Y. (1989)]. Human C region genes are readily available fromknown clones containing genes representing the two classes of L chains,the five classes of H chains and subclasses thereof.

Generally, the chimeric antibodies of the present invention are producedby cloning DNA segments encoding the H and L chain antigen-bindingregions of a specific Ab of the invention, preferably non-human, andjoining these DNA segments to DNA segments encoding human C_(H) andC_(L) regions, respectively, to produce chimeric Ig-encoding genes.

Thus, in a preferred embodiment, a fused gene is created that comprisesa first DNA segment that encodes at least the antigen-binding region ofnon-human origin, such as CDR1, CDR2 and CDR3 of the V region withjoining (J) segment, linked to a second DNA segment encoding at least apart of a human C region.

Chimeric Ab fragments, such as F(ab′)₂ and Fab, can be prepared bydesigning a chimeric H chain gene that is appropriately truncated. Forexample, a chimeric gene encoding an H chain portion of an F(ab′)₂fragment would include DNA sequences encoding the CH₁ domain and hingeregion of the H chain, followed by a translational stop codon to yieldthe truncated molecule.

One common feature of all Ig H and L chain genes and their encoded mRNAsis the J region. H and L chain J regions have different sequences, but ahigh degree of sequence homology exists (greater than 80%) among eachgroup, especially near the C region. This homology is exploited in thismethod and consensus sequences of H and L chain J regions can be used todesign oligonucleotides for use as primers for introducing usefulrestriction sites into the J region for subsequent linkage of V regionsegments to human C region segments.

C region cDNA vectors prepared from human cells can be modified bysite-directed mutagenesis to place a restriction site at the analogousposition in the human sequence. For example, one can clone the completehuman k chain C (C_(k)) region and the complete human g-1 C region(C_(g-1)). In this case, the alternative method based upon genomic Cregion clones as the source for C region vectors would not permit thesegenes to be expressed in bacterial systems where enzymes needed toremove intervening sequences are absent. Cloned V region segments areexcised and ligated to L or H chain C region vectors. Alternatively, thehuman C_(g-1) region can be modified by introducing a termination codonthereby generating a gene sequence that encodes the H chain portion ofan Fab molecule. The coding sequences with linked V and C regions arethen transferred into appropriate expression vehicles for expression inappropriate hosts, prokaryotic or eukaryotic.

In another aspect of the invention, the targeting molecule is arelatively small molecule such as a straight chain or cyclicoligopeptide or peptidomimetic having a molecular weight of about 400 toabout 1000 amu. One illustrative cyclic oligopeptide is the cyclictetrapeptide referred to as cyclic-(RGDyK) that binds to the a_(v)b₃receptor expressed on some tumors and on the endothelial cells of tumorneovasculature [Yapp et al., Mol Imaging June 2013 12(4):263-272].

A peptidomimetic is a compound whose essential elements (pharmacophore)mimic a natural peptide or protein in 3D space and retain the ability tointeract with the biological target and produce the same biologicaleffect as the natural peptide or protein [Vagner et al., Curr Opin ChemBiol June 2008 12(3):292-296]. An illustrative peptidomimetic ofinterest here is an inhibitor of prostate-specific membrane antigen(PSMA).

PSMA is a surface type 2 integral membrane glycoprotein with folatehydrolase and carboxypeptidase, and internalization activities[Cimadamore et al., Front Oncol Dec. 21, 2018 8:article 653]. PSMA ishighly expressed in prostate cancer tumor cells as well as vessels ofvarious non-prostatic solid tumors.

Monoclonal antibody J591 and the three other anti-PSMA monoclonals areeach mouse monoclonals. One or more of those mAbs is the subjectdisclosed and/or claimed in the following U.S. Pat. Nos. 6,107,090;6,136,311; 6,649,163; 6,770,450; 7,045,605; 7,112,412; 7,163,680;7,192,586; 7,514,078; 7,666,414; 7,666,425; and 8,951,737.

Structural and functional homology between N-acetylaspartylglutamatepeptidase (N-acetylated-alpha-linked acid dipeptidase;NAAALDASE) hasbeen identified as have several inhibitors for NAAALDASE. One of themost advanced peptidomimetic inhibitors are urea-based PSMA ligands,which usually consist of 3 components: a binding motif(glutamate-urea-lysine [Glu-urea-Lys]), a linker and aradiolabel-bearing moiety (chelator molecule for radiolabeling). Aparticularly useful such molecule is illustrated below without thechelator.

Illustrative of branched oligosaccharide targeting species are thesialyl-Lewis a (sLe^(a)) sialyl-Lewis x (sLe^(x)) antigens that bind toselectin receptors on endothelial cells and others that can lead tometastisis. The anti-CD33 mAb, lintuzumab, binds to the sialoadhesinreceptor CD33.

Folic acid and derivatives can be used as a targeting species forcancerous kidney cells that overexpress the folic acid receptor. Thefolate receptor is also overexpressed in cancers of the brain, kidney,lung, ovary, and breast relative to lower levels in normal cells (see,e.g., Sudimack et al., Adv Drug Deliv Rev 2000 41:147-162). Figliola etal., RSC Adv 2019 9:14078-14092, provides a synthetic route forpreparation of folate targeting species with the drug prodigiosene usingseveral α,ω-amino linking groups such as ethylene diamine, an ethyleneoxide, cystamine, and a diamino oligo oxytheylene.

Immune cells have an affinity for mannose, and several RGD-containingtargeting peptides that contain 4 to 30 amino acid residues are knownand many are described by Beer et al., Methods Mol. Biol. 2011680:183-200; Beer et al., Theranostics 2011 1:48-57; Morrison et al.,Theranostics 2011 1:149-153; Zhou et al., Theranostics 2011 1:58-82; andby Auzzas et al., Curr. Med. Chem. 2010 17:1255-1299, and Goonewardenaet al., U.S. Pat. No. 9,931,412.

Pharmaceutical Compositions

A pharmaceutical composition containing a theranostic effective amountof a contemplated targeted radiopharmaceutical dissolved or dispersed ina pharmaceutically acceptable diluent is utilized in a contemplatedmethod. In one embodiment, a theranostic effective amount is a targetedcell-killing effective amount as the treatment is therapeutic. Such acomposition is administered in vivo into in a mammalian host animal tobind to and kill unwanted targeted cells such as cancer cells andaberrant immune cells.

Illustrative unwanted targeted cells include cells associated withundesired cell migration, invasion, proliferation, immune response orangiogenesis. Illustrative of such cells are aberrant immune cells and,cancer cells such as those of lung cancer, ovarian cancer, prostatecancer, brain cancer, bladder cancer, head and neck cancer, pancreaticcancer and colon cancer. Treatment of blood cancers such as acutemyeloid leukemia that express the CD33 marker, and breast cancers thatexpress the HER2 marker is also contemplated.

A theranostic amount of targeted radiopharmaceutical Q⁺³ ionadministered therapeutically to provide a targeted cell-killingeffective amount usually varies with the patient and the severity of thedisease such a tumor load in cancer situations that the patient has.However, two to about four cycles of about 80 to about 120 kBq/kg bodyweight every other month (bimonthly; at about 60-day intervals)typically shows positive results. The use of three cycles of about 100kBq/kg body weight with the same administration regimen was reported toprovide positive results using ²²⁵AC-PSMA-617 that utilizes a DOTA-basedchelating agent in prostate cancer patients leading to completeremissions in some patients. See, Kratochwil et al., J Nucl Med 201657(12):1941-1944; Langbein et al., J Nucl Med 2019 60:13S-19S; and Ederet al., Pharmaceuticals 2022 15:267. Such dosages can be used to providea basis for dosages for therapeutic treating of other conditions.

For diagnostic purposes, the host is administered a theranostic amountthat is a target cell-binding (diagnostic) effective amount of thetargeted radiopharmaceutical. The host is thereafter maintained for atime period of about 1 hour to several days, more usually about 1 toabout 4 hours, for the radiopharmaceutical to bind to the targetedcells. The maintenance times can depend on several factors such as thedecay rate of the trivalent isotope used and the clearance rate of thetargeted radiopharmaceutical. The maintained host mammal is thereafterscanned as by a PET scan for positron emissions (PET scan) or by a gammaray detector (e.g., SPECT scan) to detect and locate the radiationemitted by the target cell-bound targeted radiopharmaceutical, andthereby identify one or more of the following 1) that targeted cellswere present in the host, 2) the location in the host body of thetargeted cells, 3) the size and possibly 4) the shape of the mass ofcells bound by the targeting species.

The diagnostically-effective amount of targeted radiopharmaceuticaladministered is typically enough radioisotope to provide about 0.5 toabout 6 mCi for an adult, and appropriately less for a child. In-111 istypically used at about 111 MBq (3 mCi) to about 222 MBq (6 mCi) forintravenous administration to an average adult (70 kg). Patients canreceive Zr-89 at about 0.5 to about 2 mCi by intravenous administrationfor a whole-body PET scan.

Because a contemplated targeted radiopharmaceutical pharmaceuticalcomposition is intended for parenteral administration as by injection,such a composition should contain an electrolyte, and preferably haveapproximately physiological osmolality and pH value of the mammalianspecies intended as the recipient. A preferred concentration of singlycharged electrolyte ions in a targeted radiopharmaceuticalpharmaceutical composition is about 0.5 to about 1.5% (w/v), morepreferably at about 0.8 to about 1.2% (w/v), and most preferably at aconcentration of about 0.9% (w/v). The about 0.9% (w/v) concentration isparticularly preferred because it corresponds to an approximatelyisotonic solution for a human. In a further preferred embodiment, theelectrolyte in a chemoablative pharmaceutical composition is sodiumchloride.

Electrolytes at such levels increase the osmolality of the targetedradiopharmaceutical pharmaceutical composition. Thus, as an alternativeto specifying a range of electrolyte concentrations, osmolality can beused to characterize, in part, the electrolyte level of the composition.It is preferred that the osmolality of a composition be greater thanabout 100 mOsm/kg and less that about 520 mOsm/kg, more preferably thatthe osmolality of the composition be greater than about 250 mOsm/kg, andmost preferably that it be about 300 to about 500 mOsm/kg.

It is preferred that the pH value of the targeted radiopharmaceuticalcomposition be about 4 to about 9, to yield maximum solubility of thetargeted radiopharmaceutical in an aqueous vehicle and assurecompatibility with biological tissue. A particularly preferred pH valueis about 5 to about 8, and more preferably between about 6 to about 7.5.

The pH value of the targeted radiopharmaceutical pharmaceuticalcomposition can be regulated or adjusted by any suitable means known tothose of skill in the art. The composition can be buffered or the pHvalue adjusted by addition of acid or base or the like.

Because a contemplated targeted radiopharmaceutical pharmaceuticalcomposition is intended for parenteral administration route, it isfurther preferred that it be sterile, such as required for conformanceto U.S. Pharmacopeia (USP) <71>, and further that it contains negligiblelevels of pyrogenic material, such that it conforms to USP <85> (limulusamebocyte lysate assay) or to USP <151> (rabbit pyrogen test), or tosubstantially equivalent requirements, at a pyrogen or endotoxin levelequivalent to not more than (NMT) 10 endotoxin units (EU) per mL.Moreover, the pharmaceutical composition should conform to requirementslimiting content of particulate matter as defined in USP <788> (i.e.,NMT 3000 particulates greater than 10 microns in size, and NMT 300particulates greater than 25 microns in size, per container) orsubstantially equivalent requirements. Each of these references from theUSP is incorporated herein by reference.

Illustrative mammalian animal hosts to which a contemplated targetedradiopharmaceutical composition can be administered include a primatesuch as a human, an ape such as a chimpanzee or gorilla, a monkey suchas a cynomolgus monkey or a macaque, a laboratory animal such as a rat,mouse or rabbit, a companion animal such as a dog, cat, horse, or a foodanimal such as a cow or steer, sheep, lamb, pig, goat, llama or thelike.

A contemplated pharmaceutical composition is usually administered aplurality of times to a mammalian host over a period of weeks, ormonths. As noted, a usual administration regimen is carried out everyother month. Screenings of the host between administrations providesupdates from which an attending physician can make determinationsconcerning further treatments. As noted before, a series of threebimonthly (about 60-days apart) administrations of a composition of adifferent Ac-225-containing targeted radiopharmaceutical pharmaceuticalat 100 kBq/kg each produced complete remissions in some prostate cancerpatients.

Formulation of parenteral compositions is discussed in, for example,Hoover, John E., Remington's Pharmaceutical Sciences, Mack PublishingCo., Easton, Pa.; 1975 and Liberman, H. A. and Lachman, L., Eds.,Pharmaceutical Dosage Forms, Marcel Decker, New York, N.Y., 1980.

For injectable preparations, for example, sterile injectable aqueoussuspensions can be formulated according to the known art using asuitable dispersing or wetting compound and suspending materials. Thesterile injectable preparation can also be a sterile injectable solutionor suspension in a nontoxic parenterally acceptable diluent or solvent,for example, as a solution in 1,3-butanediol. Among the acceptablevehicles and solvents that can be employed are aqueous liquids atambient temperature such as water, Ringer's solution, and isotonicsodium chloride solution, phosphate-buffered saline. Liquidpharmaceutical compositions include, for example, solutions suitable forparenteral administration. Sterile water solutions of targetedradiopharmaceutical or sterile solution of the targetedradiopharmaceutical in solvents comprising water, ethanol, DMSO orpropylene glycol are examples of liquid compositions suitable forparenteral administration.

Sterile solutions can be prepared by dissolving the targetedradiopharmaceutical component in the desired solvent system, and thenpassing the resulting solution through a membrane filter to sterilize itor, alternatively, by dissolving the sterile compound in a previouslysterilized solvent under sterile conditions.

EXAMPLES Example 1

Two bifunctional chelators were purchased from Macrocyclics, Dallas,Tex. The structure of the two are shown below. The Macrocyclics catalognames them as:S-2-(4-isothiocyanatobenzyl)-1,4,7,10-tetraazacyclododecane tetraaceticacid and3,6,9,15-tetraazabicyclo[9.3.1]pentadeca-1(15),11,13-triene-4-S-(4-isothiocyanatobenzyl)-3,6,9-triaceticacid. They are also named p-SCN-Bn-DOTA and p-SCN-Bn-PCTA, respectively,as unreacted precursors. Once reacted as with a targeting species, theyare more simply referred to as DOTA and PCTA, and are so named herein.

Conjugation reactions with a monoclonal antibody (MNPR-101) wereperformed in metal-free vials and glassware was acid washed to removepotential metal contamination. Reactions were performed with 2 mg ofantibody and increasing molar reactant ratios of bifunctional chelatingagents.

Monoclonal antibody (mAb) MNPR-101 is a humanized version of mouse IgG1,k, mAb ATN-658 having ATCC Accession Number PTA-8191 that is disclosedand claimed in U.S. Pat. No. 8,101,726. The mAb MNPR-101 paratope aminoacid residue sequence (CDR; complementarity determining region) is thesame as that of ATN-658, whereas the framework portions of the variableregion are humanized and the Fc portion is that of a human IgG1antibody.

For PCTA chelators, the molar reaction ratios were 1, 3, 5, 10 and 20.For the DOTA chelators the molar reaction ratios were 3, 10, 25, 50 and100. The pH of the solutions was adjusted to 9.2 with 1 M Na₂CO₃.Reactions were run at 37° C. for 1.5 hours.

Bio-Rad 10DG gravity-fed columns with a 6,000 Dalton molecular weightcut-off were used purify the conjugates. The columns were rinsed with 15mL of 0.1 M HEPES buffer in 0.1M NaCl. The pH of the buffer was 7.3. Thetotal contents of the reaction vials were introduced to the top of thecolumn and collected in 2 mL tubes. Multiple 0.5 mL elutions with thesame buffer were also captured in separate tubes. The UV absorbance at280 nm of each fraction was measured to determine the fractionscontaining protein. Typically, protein eluted in 4 fractions that werecombined. The protein content of the combined fractions was measuredusing a Pierce BCA assay kit. The concentrations of the proteinconjugates produced was about 1 mg/mL.

Analysis of each conjugate was performed with size exclusion HPLC. Thecolumn was from IGM Tosoh (TSKgelG3000SWx1; Tosoh Bioscience LLC, Kingof Prussia, Pa.). The mobile phase was phosphate-buffered saline and theflow rate was 1 mL/minute. A UV detector at 280 nm was used. HPLCresults showed an early-eluting peak with about an 8-minute retentiontime consistent with high purity conjugates. The retention time of theconjugates decreased slightly with higher ratios of bifunctionalchelators consistent with the addition of chelants to the antibody.

Example 2

Conjugates were prepared by the method of Example 1 but with a chelantto protein molar reaction ratios of 12 and 25. Typical reaction yieldsare about 30%. Thus, average CAR numbers of about 4 and 8 are expectedfor the reactions.

Ac-225 was obtained from ORNL. The reaction vial contained solid Ac-225which was dissolved using 0.2 M HCl. The same 4 conjugates as describedin the previous section were used to prepare Ac-225 chelates. A ratio of50 μCi of Ac-225 to 50 μg MNPR-101-PCTA chelant conjugate was used suchthat if there were a 100% yield, the specific activity would be 1mCi/mg. Reactions were run in 100 μL volumes. That volume included about4 μL Ac-225 in 0.2M HCl, 60 μL 0.1 M ammonium acetate buffer, and 36 μLMNPR-101-PCTA or -DOTA conjugate. Reactions were incubated at pH 5.8 and37° C. for 60 minutes.

The radiochemical yield of the reactions was determined by diluting a 50μL aliquot of reaction to 3 mL in buffer and passing through a 30 kDaAmicon® filter. Small, non-chelated Ac-225 ions pass through the filter,whereas the conjugate is retained by the filter. Samples were counted ona Ge detector after 45 minutes using the first daughter of Ac-225(Fr-221). In addition, samples were counted using a dose calibratorafter overnight (about 18 hours) equilibration of Ac with its daughters.The results of the chelation are shown below.

GE Detector (Counts) Dose Calibrator (μCi) Yield Yield Conjugate ProductFiltrate (%) Product Filtrate (%) PCTA 12:1 14572  153 99.0 12.17 0.1598.8% PCTA 25:1 12434   37 99.7% 11.95 0.03 99.7% DOTA 12:1  2985 1075821.7  2.72 9.88 21.6% DOTA 25:1  4141 9965 29.4  3.51 9.02 28.0%

The table above shows quantitative yields for both the PCTA conjugateswhereas the DOTA conjugates have much lower yields and there is asignificant difference between the 12:1 and 25:1 conjugates.Surprisingly, even at low CAR numbers, the PCTA conjugates exhibit highyields.

The specific activity of the chelates formed from PCTA was 1,000 μCi/g,whereas the specific activity for the chelates from conjugates preparedfrom DOTA ranged from about 216 to 284 mCi/g. This head-to-headcomparison between DOTA and PCTA shows the superiority of the PCTAchelators compared to DOTA chelators for chelating Ac.

High Performance Liquid Chromatography (HPLC) using a size exclusioncolumn with phosphate-buffered saline as a mobile phase was used todetermine the purity of the above samples. The HPLC data gavepractically the same results as the filtration method.

Example 3

The MNPR-PCTA conjugate of Example 2 with a chelant to antibody startingreaction ratio of 12:1 was chelated with Ac-225. This would producespecific activity of 1 mCi/mg if the reaction were quantitative. Thesame chelation reaction was performed with the DOTA conjugate ofMNPR-101 using a 25:1 chelant to antibody molar reaction ratio. Inaddition, bovine serum albumin with no chelants added was used as anegative control.

The total volume of each of the reactions was 150 μL. Each of thereactions was measured for yield using the filtration method of Example2. The percent of the activity in the retentate was used as the yield ofthe reaction.

In parallel studies, the above reactions were carried out furthercontaining 35 μL of 0.1M diethylenetriamine pentaacetate (DTPA) and thereactions stood at room temperature for one hour. At this time, thefiltration method using counts as above was again used to determine theyield or purity. The results of both studies are shown in the Tablebelow.

Reaction Yield After DTPA Challenge Test Material Yield Yield (CAR)Product Filtrate (%) Product Filtrate (%) MNPR-PCTA(12) 8013 72 99.16746 131 98.1 BSA 462 12938 3.4 142 10999 13 MNPR-DOTA(25) 1150 117308.9 706 11945 5.6

MNPR-PCTA(12) had an initial yield of 99.1%. After DTPA challenge, thechelate lost only about 1% of the activity. In contrast, the MNPR-DOTA(25) only had an initial yield of 8.9% and that decreased to 5.6% afterthe DTPA challenge. In addition, the control BSA only showed 3.4% of theactivity associated with the protein (non-specific binding) decreasingto 1.3% after DTPA wash. The data are consistent with PCTA outperformingDOTA in the ability to chelate Ac-225 even at a lower CAR ration. Inaddition, the lack of binding with naked BSA shows that non-specificbinding is not an issue.

Example 4

The conjugate between MNPR-101 (MNPR) and PCTA has been shown toefficiently chelate Ac-225. In a head-to-head comparison, Ac-225chelated much more efficiently to the PCTA conjugate than with the DOTAconjugate.

Ac-225 was obtained from ORNL. The conjugates used for these reactionswere previously prepared and described in Examples 2 and 3, above.Bovine serum albumin (BSA) was used as a negative control proteinwithout any chelators attached to it. MNPR-PCTA(12) refers to theMNPR-101 conjugate made with PCTA with a starting molar reaction ratioof 12:1 p-SCN-Bn-PCTA (PCTA) to antibody. MNPR-DOTA(25) refers to theconjugate of MNPR-101 with a starting molar reaction ratio of 25p-SCN-Bn-DOTA (DOTA) chelators to antibody.

Reactions were targeted to produce 1 mCi/mg assuming 100% incorporationof the Ac into the antibody. The reactions were run in 150 μL volumesand incubated at pH 5.8 and 37° C. for 60 minutes. Following thereaction, 35 μL aliquots of each reaction were mixed with 35 μL of 1Mdiethylenetriamine pentaacetate (DTPA) and allowed to stand at roomtemperature for 1 hour.

The solutions were tested for the percent Ac-225 associated with theprotein by filtration (counts) as described above as a function of time(1, 24 and 72 hours). The results of the initial study are shown in thetable below as the percent Ac-225 associated with the protein as afunction of time.

1 hour 24 hours 72 hours Test Material in DTPA in DTPA in DTPAMNPR-PCTA(12)  99%  98%  73% BSA 3.4% 1.3% 3.2% MNPR-DOTA(25) 8.9% 5.6%6.6%

The percentage designates the relative amount of activity in the filtercompared to the total (filter+filtrate). MNPR-PCTA(12) gave the bestresults with 99% and 98% attached to the antibody (on the filter) after1 and 24 hour incubation with DTPA. The purity dropped to 73% after 72hours. Note that there is no radioprotectant added and Ac-225 gives ahigh radiation dose to the solution. The fact that the isotope remainedassociated with the protein shows a high degree of stability, and thatthere was little loss of the bismuth decay product to the solutionduring that time periods examined.

Both the control (BSA) and MNPR-DOTA(25) have significantly lowerpercentages of the activity associated with the protein. High resolutiongamma spectroscopy analysis of the solutions was consistent with thefiltration results in Table 1.

The antibody MNPR-101 conjugated with PCTA with a starting chelator toantibody molar reaction ratio of 12:1 was shown to reproducibly chelateAc-225 in high yield and high specific activity (1,000 μCi/mg).Incubation of the material in excess DTPA showed a high degree ofstability even when the formulation did not contain any radioprotectant.A head-to-head comparison with the same antibody conjugated with DOTAwith a starting ratio of 25:1 ligand to protein molar ratio gave muchlower yields showing the advantage of PCTA over DOTA for chelatingAc-225. Naked BSA was used as a control showing low amounts ofnon-specific binding.

Example 5

A targeted radiopharmaceutical containing Ac-225 chelated by PCTA bondedto mAb MNPR-101 as illustrated by Formula I, where Q⁺³, is ¹²⁵Ac⁺³, wasprepared as described earlier. The starting molar ratio of chelator toantibody was 12 to 1. Fifty μCi of Ac-225 was combined with 50 μg of theMNPR-PCTA conjugate and the pH value adjusted to 5.8 with ammoniumacetate for 60 minutes at 37 C. The total volume of the reaction was 100μL.

A volume of 25 μL of the reaction mixture was analyzed on highperformance liquid chromatography using a size exclusion column. Themobile phase was 0.1 M phosphate buffer at pH=7.4 and the flow rate was1 mL/minute. Detection was by UV absorption at 280 nm and also byradiometric detector.

Evaluation of the UV and radiometric detector showed the radioactivityco-eluting with the protein. A size exclusion column separates chemicalsbased on size. Because most of the radioactivity from a solution ofAc-225 comes from its radioactive daughters, we would expect radioactivemetals that are not attached to the protein to elute at a later time.There was no radioactive signal with retention times consistent withsmall molecules.

This result is consistent with the MNPR-101-PCTA conjugate chelatingradioactive Ac-225 daughters such as Bi-213. Not to be bound by theorybut the excellent binding properties of the PCTA conjugate are believedto be a result of the chelator binding not only Ac-225 but daughterssuch as Bi-213 being trivalent and/or not radioactive.

Bismuth ions can form very insoluble compounds that could precipitatecarrying both bismuth and actinium ions. Prevention of bismuth compoundsprecipitation by the mAb-linked-PCTA chelating functionality providesanother benefit of this invention.

Similar size exclusion column studies using DOTA as the chelating agentlinked to mAb MNPR-101 show different results. Thus, when DOTA is used,the on-line radiation detector shows very little signal associated withthe protein and most of the activity in later-eluting peaks that areindicative of radioactive metals that are not attached to the protein.

Example 6

PCTA conjugates were prepared with humanized mAb MNPR-101 in parallelwith two other illustrative mouse monoclonal antibodies: mAb ATN-616 andmAb ATN-292. The chelator to protein molar ratios of 12 and 75 were usedto optimize subsequent chelation of Ac-225.

MNPR-101 and ATN-616 were conjugated with PCTA at the molar reactionratio of 12:1, whereas ATN-292 was conjugated at a 75:1 excess. The pHvalues of the solutions were adjusted to 9.2 with 1 M NaH₂CO₃ and 0.2 MHCl. Reactions were run at 37° C. for 1.5 hours.

The conjugates so formed were purified using Bio-Rad 10DG gravity-fedcolumns (6,000 Dalton (Da) molecular weight cut-off) in which eachconjugate was eluted with 0.1 M ammonium acetate buffer, pH 5.77. Elutedfractions (0.5 mL) were collected in 1.5 mL metal-free tubes and weremeasured at UV absorbance 280 nm. 3 or 4 fractions were combined,depending on concentration of protein in the eluant, and re-concentratedusing Amicon® concentrators (30 kDa). Combined fractions were analyzedusing a Pierce™ BCA Assay Kit (Thermofisher; Final proteinconcentrations were about 2-3 mg/mL).

Size-exclusion high performance liquid chromatography was utilized toanalyze conjugate purity, as previously described, withphosphate-buffered saline solvent and flow rate of 1 mL/minute. HPLCresults revealed the expected about 8-minute peaks observed from thenaked antibodies and decreased retention time (Rt) of the conjugatesconsistent with addition of the bifunctional chelator PCTA.

Results suggest that the increase in retention time (ΔRt) observedbetween the conjugates and respective naked mAb(s) is related to theconjugates' subsequent ability to chelate Ac-225, in that a greater ΔRtcorrelates to a higher number of chelating agents bonded to theantibody. Differences in retention times from the three conjugates andnaked antibodies are shown below.

Retention Naked Ab Time (Rt) Ab Conjugate Protein Rt ΔRt MNPR-101 7.981MNPR-101- 7.685 0.296 PCTA (12:1) ATN-616 7.551 ATN-616- 7.472 0.079PCTA (12:1) ATN-292 8.218 ATN-292- 7.689 0.529 PCTA (75:1)

Example 7

A reaction vial containing solid Ac-225 was obtained from ORNL and wasdissolved using 0.2 M HCl. The three conjugates from Example 6 were usedto prepare Ac-225 chelates. A ratio of 100 μCi of Ac-225 to 100 μgmAb-PCTA chelant conjugate for all reactions was used such that if therewere a 100% yield, the specific activity would be 1 mCi/mg. Reactionswere run in about 110 μL volumes including approximately 10 μL Ac-225 in0.2 M HCl, 60 μL 0.1 M ammonium acetate buffer, and 40 μL mAb-PCTAconjugate, dependent upon and normalized against each proteinconcentration. Reactions were incubated at pH 5.7 and 37° C. for 60minutes.

25 μL aliquots of each chelation reaction were purified by eluting 0.5mL fractions on Bio-Rad 10DG gravity-fed columns with 0.1 M ammoniumacetate buffer. The Ac-labeled conjugate is expected to elute in 3-4“peak fractions”, which are summed against the activity remaining on thecolumn to determine radiochemical yield. After a minimum of 5 hours (topermit Ac equilibration with its daughters), the fractions andrespective columns were measured on the dose calibrator (Capintec,setting #086). Results from each chelations' gravity-fed fractionsmeasured on the dose calibrator are shown in the following tables.

MNPR-101-PCTA- ATN-616-PCTA- ATN-292-PCTA- Ac-225 (12:1) Ac-225 (12:1)Ac-225 (75:1) Yield Yield Yield Fraction μCi (%) Fraction μCi (%)Fraction μCi (%) 1 0.02 0.1 1 0.03 0.2 1 0.09 0.4 2 0.05 0.2 2 0 0.0 20.11 0.5 3 0.07 0.3 3 0.01 0.1 3 0.09 0.4 4 2.3 11.1 4 0.76 3.9 4 1.661.7 5 7.4 35.7 5 6.59 34.1 5 7.59 37.1 6 5.08 24.5 6 5.05 26.1 6 5.426.4 7 1.92 9.3 7 2.5 12.9 7 1.97 9.6 8 0.45 2.2 8 0.43 2.2 8 0.48 2.3 90.18 0.9 9 0.13 0.7 9 0.2 1.0 10 0.23 1.1 10 0.12 0.6 10 0.1 0.5 11 0.070.3 11 0.03 0.2 11 0.08 0.4 12 0.13 0.6 12 0.06 0.3 12 0.05 0.2 13 0.140.7 13 0.05 0.3 13 0.08 0.4 14 0.09 0.4 14 0.05 0.3 14 0.1 0.5 15 0.050.2 15 0 0.0 15 0.11 0.5 Column 2.55 12.3 Column 4 18.3 Column 2 11.5Total 21 Total 19 Total 20 Peak Yield: 80.6% Peak Yield: 77.0% PeakYield: 75.5%

Peak fractions from each reaction were measured at time points 24 hoursand 48 hours post-purification to determine the chelants' ability toretain Ac-225 daughter isotopes. Peak activity increasing as a functionof time provides evidence that the chelants did not effectively controlthe daughter isotopes. However, if activity decreased at a rateconsistent with Ac-225 degradation, evidence suggests that the chelantswere able to retain Ac-225 and its daughters.

Results observed in the tables below provide evidence of the threechelating systems effectively controlling Ac-225 daughters, as each peakactivity does not exhibit a radioactivity increase as a function oftime.

ATN 616-PCTA-Ac-225 (12:1) Fraction μCi 24 hr (μCi) 48 hr (μCi) 5 8.59NT* 6.25 6 5.05 NP 5.02 7 2.5 NT* 2.43 *NT = Not Tested

ATN-292-PCTA-Ac-225 (75:1)1 Fraction μCi 24 hr (μCi) 48 hr (μCi) 5 7.597.49 7.1 6 5.4 5 25 4.97 7 1.97 1.87 1.79

MNPR-101-PCTA-Ac-225 (12:1) Fraction μCi 24 hr (μCi) 48 hr (μCi) 5 7.47.4 6.93 s 5.03 5.05 4.79 7 1.92 1.78 1.78

Example 8

20 μL samples of each chelation reaction were analyzed via HPLC using anisocratic method (1×PBS solvent, pH 7.4) with detection method via UVabsorption at 280 nm and radiometric detector. 1 mL fractions werecollected per minute and permitted to equilibrate (>5 hours) then weremeasured on a NaI detector with a wide window.

As observed in Example 5, HPLC results showed the radioactivityco-eluting with the proteins from the three reactions. There was noradioactive signal with a retention time consistent with a smallmolecule, further supporting the inference that the PCTA chelator bindsAc-225 and its daughters. The results of each reaction yield are shownin the table below:

Nal Detector, Counts Per Minute Test Article Reaction Yield (%) MNPR101-PCTA-Ac-225 96.2% ATN-616-PCTA-Ac-225 92.7% ATN-292-PCTA-Ac-22597.8%

Although the Peak Yields of the three reactions when analyzed by thedose calibrator show 80.6%, 77.0%, and 75.5%, respectively, as describedin Example 7, these same reactions show Reaction Yields of 96.2%, 92.7%,and 97.8%, respectively, when analyzed using HPLC purification and NaIdetection. This variance is understood to stem from a lack of theability to measure activity remaining on the size-exclusion column, thusobserving the more conservative yields from the Bio-Rad gravity-fedcolumns and dose calibrator values.

The methods and results described suggest the bifunctional chelator inquestion, PCTA, shows remarkable ability to bind Ac-225 not only withhumanized mAb MNPR-101, but also when linked to other antibodies aswell, such as the two mouse monoclonal antibodies mAb ATN-616 and mAbATN-292.

Example 9

Humanization of Variable (V) Region Amino Acid Residue Sequence of MousemAb ATN-658 The consensus amino acid sequence (single-letter code) ofthe light chain variable region (V_(L)) and heavy chain variable region(V_(H)) polypeptides of mAb ATN-658 are set out in U.S. Pat. No.8,191,726 to Parry and Mazar, will not be repeated here and areincorporated by reference. cDNA was prepared from total RNA extractedfrom the hybridoma expressing ATN-658 and the variable regions werecloned, amplified and sequenced using standard techniques.

Following the course set out by Studnicka et al., above, human V Kappalight chain subgroup 2 (VK2) and human heavy chain subgroup 1 (VH1)consensus sequences were utilized. The cognate mouse signal sequenceswere retained.

Two sequences for each of the light chain and the heavy chain variableregions were prepared. One sequence for each chain contained only lowrisk changes and the other sequence that contained both the low risk andthe moderate risk changes were prepared for the VK2 and VH1 regions,providing a total of four sequences. Ten low risk and 1 moderate riskchanges were introduced into the light chain framework sequences and 11low risk and 5 moderate risk changes were introduced into the heavychain framework sequences. Low risk residue position changes, thoseexposed to solvent but not contributing to antigen binding or antibodystructure, are likely to decrease immunogenicity with little or noeffect on binding affinity.

The amino acid residue sequences were sent to Blue Heron Biotech LLP,(Bothell, Wash.) for codon (Chinese Hamster Ovary cells) and expressionoptimization. The optimized DNA sequences were received and sent back toBlue Heron for gene synthesis.

Transient Expression Vector Construction

Codon- and expression-optimized low risk and low plus moderate riskHuman Engineered™ light chains and heavy chains were cloned in-frameinto XOMA's proprietary transient antibody expression vectors thatcontain human Kappa and Gamma-1 constant region modules. The DNAsequences were verified (at ELIM Biopharmaceuticals, Inc., Hayward,Calif.) prior to initiating expression.

Production of Human Engineered™

ATN-658 Antibodies

The four HE™ ATN-658 variants (referred to as HE™ ATN-1, HE™ ATN-2, HE™ATN-3 and HE™ ATN-4) were produced by transient transfection in HEK293Ecells. XOMA's transient transfection approach is described in detail ina poster presented at the 2005 ASCB Annual Meeting.

Briefly, the light and heavy chains were co-transfected into XOMA'ssuspension-adapted HEK293E cells grown in IS293 medium (IrvineScientific, Irvine, Calif.) using 2 liter shake flasks. After 24 hoursin shake flasks, 200 ml of transfected cells were centrifuged,resuspended in 40 ml of fresh medium and transferred to Integra flasks(Wilson Wolf Manufacturing, Inc., New Brighton, Minn.) for production.After incubation for seven days, the cell suspensions were removed fromthe Integra flasks, centrifuged and the culture supernatants retained.Antibodies in the culture supernatants were purified on protein A spincolumns (Pro-Chem), dialyzed against PBS, concentrated and sterilefiltered.

The variable region constituent sequences of those four antibodies areillustrated in Table 1, below.

TABLE 1 Risk Level Antibody Heavy Light % Human Variant Chain Chain (asIgG1)* HE ™ ATN-1 Low Low 95.2 HE ™ ATN-2 Low Low + 95.2 Moderate HE ™ATN-3 Low + Low 95.8 Moderate HE ™ ATN-4 Low + Low + 95.8 ModerateModerate *Low or Low + Moderate Risk changes and conservation betweenmouse and human V regions wherein mouse amino acid residues arerepresented at any given position in at least two Kabat human V regionsfrom matching subtype.

Concentration was determined by A280 using an extinction coefficient of1.52. The proteins were analyzed for purity by SDS-PAGE (4-20%) and forendotoxin using an LAL assay. Purification results demonstrate that allof the antibody preparations had concentrations ≥1 mg/ml, were >90% pureand had low levels of endotoxin (<1 EU/mg).

Evaluation of Affinity of Human Engineered™

ATN-658 Antibodies by Biacore Assay Method

Kinetics analysis of mouse monoclonal antibody ATN-658 and HumanEngineered™ ATN-658 variant antibodies was conducted on a Biacore 2000®surface plasmon resonance instrument analyzer (Uppsala, Sweden) toproduce sensograms based on the antibody-surface interactions. Kineticdeterminations were performed using a capture method.

Mouse parental mAb ATN-658 was diluted in PBS to 2 mg/mL and injectedover a rabbit anti-mouse capture surface. The HE™ variants were dilutedto 1 mg/mL and injected over a protein A/G surface. Antibody injectionswere optimized to produce antibody densities of 100-200 RU.

Six serial 3-fold dilutions of soluble UPAR (suPAR) were prepared inrunning buffer (PBS), and each dilution was injected in triplicate inrandom order at 25° C. Buffer injections were evenly distributedthroughout the run. The sample injections were double-referenced againstthe blank flow cells and buffer injections to correct for any bulk shiftor non-specific binding. Data were analyzed with BiaEvaluation softwarefrom Biacore®. Sensorgrams were fit utilizing a 1:1 Langmuir model.

Humanized mAb MNPR-101

As compared to mAb ATN-658, one residue was changed in one CDR of eachof the VK2 and VH1 regions in mAb MNPR-101 as compared to the CDRsequences of mAb ATN-658 (CDR L1 and CDR H2) in arriving at the six CDRsof mAb MNPR-101. The complementarity-determining regions (CDRs) for eachvariable region that are present in paratropic regions of mAb MNPR-101and are set out in Table 2, below.

TABLE 2 Characteristics of CDRs of MNPR-101 L and H Chains No. of SEQ IDCDR* Residues Sequence1 NO CDR L1 16 RSSQSLLDSDGKTYLN 3 CDR L2 7 LVSKLDS4 CDR L3 9 WQGTHFPLT 5 CDR H1 10 GYSFTSYYMH 10 CDR H2 17EINPYNGGASYNQKIQG 11 CDR H3 10 SIYGHSVLDY 12 *CDR L1: first CDR of Lchain; CDR H2: 2^(nd) CDR of H chain, etc.

Sequences

Sequences for the VL and VH as well as the CL and CH regions of the Fabportion of mAb MNPR-101, and also the low risk sequences of the variableregions of both chains (HE™ ATN-1) are shown below.

SEQ ID NO: 1  [mAb MNPR-101 VL]Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu Ser ValThr Ile Gly Glu Pro Ala Ser Ile Ser Cys Arg Ser SerGln Ser Leu Leu Asp Ser Asp Gly Lys Thr Tyr Leu AsnTrp Leu Leu Gln Lys Pro Gly Gln Ser Pro Gln Arg LeuIle Tyr Leu Val Ser Lys Arg Asp Ser Gly Val Pro AspArg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr LeuLys Ile Ser Arg Val Glu Ala Glu Asp Val Gly Val TyrTyr Cys Trp Gln Gly Thr His Phe Pro Leu Thr Phe GlyGln Gly Thr Lys Leu Glu Ile Lys SEQ ID NO: 2 [HE™ ATN-1 VL]Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu Ser ValThr Ile Gly Glu Pro Ala Ser Ile Ser Cys Arg Ser SerGln Ser Leu Leu Asp Ser Asp Gly Lys Thr Tyr Leu AsnTrp Leu Leu Gln Lys Pro Gly Gln Ser Pro Lys Arg LeuIle Tyr Leu Val Ser Lys Arg Asp Ser Gly Val Pro AspArg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr LeuLys Ile Ser Arg Val Glu Ala Glu Asp Val Gly Val TyrTyr Cys Trp Gln Gly Thr His Phe Pro Leu Thr Phe GlyGln Gly Thr Lys Leu Glu Ile Lys SEQ ID NO: 3 [mAh MNPR-101 CDR L1]Arg Ser Ser Gln Ser Leu Leu Asp Ser Asp Gly Lys Thr Tyr Leu AsnSEQ ID NO: 4 [mAh MNPR-101 CDR L2] Leu Val Ser Lys Arg Asp SerSEQ ID NO: 5 mAh [MNPR-101 CDR L3] Trp Gln Gly Thr His Phe Pro Leu ThrSEQ ID NO: 6 [LC Signal Sequence] MSPAQFLFLL VLWIRETNG SEQ ID NO: 7[mAh MNPR-101 LC Constant Region Sequence]RTVAAPSVFI FPPSDEQLKS GTASVVCLLN NFYPREAKVQWKVDNALQSG NSQESVTEQD SKDSTYSLSS TLTLSKADYEKHKVYACEVT HQGLSSPVTK SFNRGEC SEQ ID NO: 8[mAb MNPR-101 low + Moderate Risk-VH]Glu Val Gln Leu Val Gln Ser Gly Pro Glu Val Lys LysThr Gly Ala Ser Val Lys Ile Ser Cys Lys Ala Ser GlyTyr Ser Phe Thr Ser Tyr Tyr Met His Trp Val Arg GlnAla His Gly Gln Gly Leu Glu Trp Ile Gly Glu Ile AsnPro Tyr Asn Gly Gly Ala Ser Tyr Asn Gln Lys Ile GlnGly Arg Ala Thr Phe Thr Val Asp Thr Ser Thr Ser ThrAla Tyr Met Glu Phe Ser Ser Leu Arg Ser Glu Asp ThrAla Val Tyr Tyr Cys Ala Arg Ser Ile Tyr Gly His SerVal Leu Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser SEQ ID NO: 9[HE™ ATN-1 VH] Glu Val Gln Leu Val Gln Ser Gly Pro Glu Val Val LysThr Gly Ala Ser Val Lys Ile Ser Cys Lys Ala Ser GlyTyr Ser Phe Thr Ser Tyr Tyr Met His Trp Val Lys GlnAla His Gly Gln Gly Leu Glu Trp Ile Gly Glu Ile AsnPro Tyr Asn Gly Gly Ala Ser Tyr Asn Gln Lys Ile LysGly Arg Ala Thr Phe Thr Val Asp Thr Ser Thr Arg ThrAla Tyr Met Glu Phe Ser Ser Leu Arg Ser Glu Asp ThrAla Val Tyr Tyr Cys Ala Arg Ser Ile Tyr Gly His SerVal Leu Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser SerSEQ ID NO: 10 [mAb MNPR-101 CDR H1]Gly Tyr Ser Phe Thr Ser Tyr Tyr Met His SEQ ID NO: 11[mAb MNPR-101 HC CDR H2]Glu Ile Asn Pro Tyr Asn Gly Gly Ala Ser Tyr Asn Gln Lys Ile Gln GlySEQ ID NO: 12 [mAb MNPR-101 HC CDR H3]Ser Ile Tyr Gly His Ser Val Leu Asp Tyr SEQ ID NO: 13[mAb MNPR-101 HC Signal Sequence] MGWIWIFLFL LSGTAGVHS SEQ ID NO: 14[mAb MNPR-101 HC Constant Region Sequence]ASTKGPSVFP LAPSSKSTSG GTAALGCLVK DYFPEPVTVSWNSGALTSGV HTFPAVLQSS GLYSLSSWT VPSSSLGTQTYICNVNHKPS NTKVDKRVEP KSCDKTHTCP PCPAPELLGGPSVFLFPPKP KDTLMISRTP EVTCVVVDVS HEDPEVKFNWpharmaceutical compositionsYVDGVEVHNA KTKPREEQYNSTYRVVSVLT VLHQDWLNGK EYKCKVSNKA LPAPIEKTISKAKGQPREPQ VYTLPPSREE MTKNQVSLTC LVKGFYPSDIAVEWESNGQP ENNYKTTPPV LDSDGSFFLY SKLTVDKSRWQQGNVFSCSV MHEALHNHYT QKSLSLSPGK

A SalI restriction site was placed in frame and up-stream of the encodedN-terminus of each of the heavy and light chains and a XhoI site wasinserted in frame and down-stream from the encoded C-terminus of eachchain for insertion of coding nucleic acids into their expressionvectors.

MNPR-101 Production

The heavy and light chain of the monoclonal antibody candidate werepackaged in a pUC19 plasmid. cDNA inserts encoding the monoclonalantibodies were cloned out and heavy and light chains were inserted intoexpression vectors.

After confirmation of the sequences, the DHFR-deficient CHO cell lineDUX B11 was transfected with light chain and heavy chain containingvectors and a cationic liposome mixture (Lipofectamine® 2000; InvitrogenCorp., Carlsbad, Calif.). Forty-eight hours after transfection, cellswere subcloned in 96 well dishes using a purine-free growth medium inthe presence of geneticin (G418) and 20 nM methotrexate (MTX).

After selection, all subclones were screened using a hIgG Bethyl ELISAkit. Three vials were frozen down for each of the 12 best subclones. Thetop 6 best producing subclones were then transferred to a mediumsupplemented with increasing amounts of methotrexate (MTX), an inhibitorof DHFR. MTX concentrations were sequentially increased from 20 to 1,000nM during the selection process and then to 1,500 nM MTX. TheMTX-resistant clones that grew out were screened by ELISA. After a firstseries of amplification, the two highest expressing population subcloneswere obtained in medium containing 1,000 nM MTX. These two clones wereamplified up to 1,500 nM MTX before being subcloned at 1,000 nM and1,500 nM MTX. These subclones are currently being expanded to 6 wellplates and will be screened by ELISA in the next few days. The top 2-3best subclones will be then expanded for the production of a ResearchCell Bank after adaptation to serum free medium.

Results

The ligand-binding kinetics of mouse mAb ATN-658 and the above discussedHuman Engineered™ ATN-658 antibodies were measured once. The sensorgramresults of individual assays indicated that all of thetransiently-expressed antibodies displayed a similar affinity with mAbATN-658 as well as among themselves. Results for the four combinationsof two VL and two VH chains are shown below in Table 3.

TABLE 3 Antibody Variant ka kd KD (pM) ATN-658 3.7e5 1.4e−4 380 HE ™ATN-1 8.6e5 2.4e−4 280 HE ™ ATN-2 6.8e5 2.6e−4 380 HE ™ ATN-3 9.5e52.7e−4 280 HE ™ ATN-4 7.0e5 2.7e−4 390

Antibody HE™ ATN-4 was renamed MNPR-101.

Example 10

An initial study of the chelation characteristics and stability ofIn-111 using a contemplated PCTA-MNPR-101 chelator-targeting species.Thus, PCTA-MNPR-101 (produced at 12:1) freshly prepared in an aqueoussolution at a pH value of 9.2 (1M NaHCO₃ and HCl) that contained 4.0mg/mL by protein analysis was incubated for 1.5 hours at 37° C. Theconjugate (220.0 mL MNPR-101-PCTA) was purified by passage through aPD10 column with elution using 0.1M ammonium acetate. Samples containingthe conjugate were collected and concentrated using a 30 kDa Amicon®concentrator (4000 rpm for 20 minutes).

Three aqueous chelation reactions were set up, each with activity ofabout 200 μCi for a target specific activity of 10 mCi/mg. Each wasmixed with In-111 chloride obtained from BWXT Medical, Ottawa, ON,Canada. All reactions were stored at 4° C. and assayed for stabilityafter 24, 48 and 72 hours.

Stability in this context is the maintenance of radioactive ionchelation over time. Stability was determined by gravity fed SEC column(PD10 6,000 Dalton cut off), HPLC and TLC for comparison.

Three aqueous chelation reactions were set up, each with activity ofabout 200 μCi for a target specific activity of 10 mCi/mg. These were asfollows:

1) Incubated at 37° C. for 30 minutes. Stored at 4° C. for 72 hours.

2) Incubated at room temperature for 30 minutes. Stored at 4° C. for 24hours.

3) Incubated at room temperature for 1.5 hours. Stored at 4° C. for 48hours.

The results of this initial study are shown in the Table below.

Initial PD10 Final Reaction TLC Stability column HPLC TLC ReactionConditions (AVG %) (Hours) (%) (%) (Avg %) 1 37°, 30 min 90.9 72 65.387.7 94.6 2 RT, 30 min 93.1 24 84.6 83.5 87.3 3 RT, 1.5 Hr 90.3 48 66.184.5 87.3

The results of this initial study showed that relatively high yields ofchelation were obtained at 10 mCi/mg targeted specific activity.Conditions could likely be optimized to increase yields. Each of thethree different analytical methods showed that a chelate was formed.Given that the half-life of Indium-111 is about 2.8 days, reasonablechelated In-111 stability was observed.

Each of the patents, patent applications and articles cited herein isincorporated by reference. The use of the article “a” or “an” isintended to include one or more.

The foregoing description and the examples are intended as illustrativeand are not to be taken as limiting. Still other variations within thespirit and scope of this invention are possible and will readily presentthemselves to those skilled in the art.

1. A targeted radiopharmaceutical that comprises a targeting species chemically-bonded to PCTA-chelated Q⁺³ radioactive isotope ion, wherein the PCTA chelator has the general structural formula shown below in Formula I

wherein Q⁺³ is a trivalent radioactive isotope ion; six of R¹, R², R³, R⁴, R⁵, R⁶, and R⁷ are H and the seventh contains a reacted functionality, Z, that forms said chemical bond with said targeting species, T; “g” is a number whose average value is 1 to about 12 that indicates the average number of chelated PCTA-chelated trivalent radioactive ions, Q⁺³, per each molecule of targeting species, T; X¹, X², and X³, are the same or different substituent groups that can coordinate to said Q⁺³ radioactive isotope ion and/or help neutralize the ionic charge; and an optional anion, Y⁻, is optionally present in an amount needed to balance the ionic charge.
 2. The targeted radiopharmaceutical according to claim 1, wherein said reacted functionality, Z, is selected from the group consisting of one or more of a reacted Michael reaction acceptor, a reacted isocyanato group, an activated carboxyl group, and a 1,4-disubstituted-1,2,3-triazine formed by the reaction of an azide and an alkyne.
 3. The targeted radiopharmaceutical according to claim 1, wherein said targeting species, T, is selected from the group consisting of one or more of a chemically-bonded antibody or paratope-containing portion of an antibody, a chemically-bonded hormone, a chemically bonded non-antibody protein, a chemically-bonded cytokine, a chemically bonded aptamer, a chemically bonded nucleic acid or oligonucleotide, a straight chain or cyclic oligopeptide, and a straight chain or branched oligosaccharide.
 4. The targeted radiopharmaceutical according to claim 1, wherein each of said X¹, X², and X³, is a —(CH₂)_(n)CO₂M or a —PO₃M₂ substituent, n is zero or 1, and M is H⁺ or a alkali metal cation.
 5. The targeted radiopharmaceutical according to claim 4, wherein each of said X¹, X², and X³, is a —(CH₂)_(n)CO₂M substituent, n is zero or 1, and M is H⁺ or a alkali metal cation.
 6. The targeted radiopharmaceutical according to claim 5, wherein n is zero.
 7. The targeted radiopharmaceutical according to claim 1, wherein Q⁺³ is an Ac-225, Bi-212, Bi-213, Zr-89 or In-111 ion.
 8. The targeted radiopharmaceutical according to claim 7, wherein Q⁺³ is an Ac-225, Bi-212 or Bi-213 ion.
 9. The targeted radiopharmaceutical according to claim 7, wherein Q⁺³ is a Zr-89 or In-111 ion.
 10. The targeted radiopharmaceutical according to claim 1, wherein said targeting species, T, is a chemically-bonded antibody or paratope-containing portion of an antibody.
 11. The targeted radiopharmaceutical according to claim 10, wherein said antibody or paratope-containing portion of an antibody is a monoclonal antibody (mAb) or a paratope-containing portion thereof.
 12. The targeted radiopharmaceutical according to claim 11, wherein said monoclonal antibody or a paratope-containing portion thereof is the mAb designated ATN-658 produced by hybridoma having ATCC Accession #PTA-8191 or paratope-containing portion of ATN-658.
 13. The targeted radiopharmaceutical according to claim 11, wherein said mAb is humanized.
 14. A pharmaceutical composition that comprises a theranostic effective amount of a targeted radiopharmaceutical according to claim 1 dissolved or dispersed in a pharmaceutically acceptable diluent.
 15. The pharmaceutical composition according to claim 14, wherein said pharmaceutically acceptable diluent is an aqueous liquid at ambient temperature and is adapted for parenteral administration.
 16. The pharmaceutical composition according to claim 15, wherein said composition is isotonic to the blood of the intended mammalian species host recipient.
 17. The pharmaceutical composition according to claim 16, wherein said intended mammalian species host recipient is a human.
 18. A method for treating a mammalian host having a disease, disorder or condition characterized by undesired angiogenesis, tumor growth and/or tumor metastasis comprising administering to said host a pharmaceutical composition of claim 14 wherein said theranostic effective amount is a targeted cell-killing effective amount of said targeted radiopharmaceutical.
 19. The method according to claim 18, wherein the disease, disorder or condition is cancer.
 20. The method according to claim 19, wherein said cancer is selected from the group consisting of one or more of lung cancer, ovarian cancer, prostate cancer, brain cancer, bladder cancer, head and neck cancer, pancreatic cancer or colon cancer.
 21. The method according to claim 18, wherein said mammalian host is a human.
 22. The method according to claim 18, wherein said administration is repeated.
 23. The method according to claim 21, wherein said targeted radiopharmaceutical is administered in an amount sufficient to provide about 80 to about 120 kBq/kg body weight to said mammalian host.
 24. The method according to claim 23, wherein said administration is repeated.
 25. The method according to claim 24, wherein said administration is repeated at about 60-day intervals.
 26. A method for assaying a mammalian host having a disease, disorder or condition characterized by undesired angiogenesis, tumor growth and/or tumor metastasis comprising a) administering to said host a pharmaceutical composition of claim 14 wherein said theranostic amount is a diagnostically effective amount of said targeted radiopharmaceutical; b) maintaining said host for a time period of about 1 hour to several days for the radiopharmaceutical to bind to the targeted cells; and c) scanning the maintained host to detect and locate the radiation emitted by the target cell-bound targeted radiopharmaceutical.
 27. The method according to claim 26, wherein the disease, disorder or condition is cancer.
 28. The method according to claim 27, wherein said cancer is selected from the group consisting of one or more of lung cancer, ovarian cancer, prostate cancer, brain cancer, bladder cancer, head and neck cancer, pancreatic cancer or colon cancer.
 29. The method according to claim 28, wherein said mammalian host is a human.
 30. The method according to claim 26, wherein said administration is repeated.
 31. The method according to claim 29, wherein said targeted radiopharmaceutical is administered in an amount of about 0.5 to about 6.0 mCi to said human. 